Traumatic brain injury (TBI) results in rapid reactive oxygen species (ROS) production and oxidative damage to essential brain cellular components leading to neuronal dysfunction and cell death. It is increasingly appreciated that a major player in TBI-induced oxidative damage is the reactive nitrogen species (RNS) peroxynitrite (PN) which is produced in large part in injured brain mitochondria. Once formed, PN decomposes into highly reactive free radicals that trigger membrane lipid peroxidation (LP) of polyunsaturated fatty acids (e.g. arachidonic acid) and protein nitration (3-nitrotyrosine, 3-NT) in mitochondria and other cellular membranes causing various functional impairments to mitochondrial oxidative phosphorylation and calcium (Ca2+) buffering capacity. The LP also results in the formation of neurotoxic reactive aldehyde byproducts including 4-hydroxynonenal (4-HNE) and propenal (acrolein) which exacerbates ROS/RNS production and oxidative protein damage in the injured brain. Ultimately, this results in intracellular Ca2+ overload that activates proteolytic degradation of α-spectrin, a neuronal cytoskeletal protein. Therefore, the aim of this study was to establish the temporal evolution of mitochondrial dysfunction, oxidative damage and cytoskeletal degradation in the brain following a severe controlled cortical impact (CCI) TBI in young male adult rats. In mitochondria isolated from an 8mm diameter cortical punch including the 5 mm wide impact site and their respiratory function studied ex vivo, we observed an initial decrease in complex I and II mitochondrial bioenergetics within 3 hours (h). For complex I bioenergetics, this partially recovered by 12–16h, whereas for complex II respiration the recovery was complete by 12h. During the first 24h, there was no evidence of an injury-induced increase in LP or protein nitration in mitochondrial or cellular homogenates. However, beginning at 24h, there was a gradual secondary decline in complex I and II respiration that peaked at 72h. post-TBI that coincided with progressive peroxidation of mitochondrial and cellular lipids, protein nitration and protein modification by 4-HNE and acrolein. The oxidative damage and respiratory failure paralleled an increase in Ca2+-induced proteolytic degradation of the neuronal cytoskeletal protein α-spectrin indicating a failure of intracellular Ca2+ homeostasis. These findings of a surprisingly delayed peak in secondary injury, suggest that the therapeutic window and needed treatment duration for certain antioxidant treatment strategies following CCI-TBI in rodents may be longer than previously believed.
Extensive evidence has demonstrated an important role of oxygen radical formation (i.e. oxidative stress) as a mediator of the secondary injury process that occurs following primary mechanical injury to the brain or spinal cord. The predominant form of oxygen radical-induced oxidative damage that occurs in injured nervous tissue is lipid peroxidation (LP). Much of the oxidative stress in injured nerve cells initially begins in mitochondria via the generation of the reactive nitrogen species peroxynitrite (PN) which then can generate multiple highly reactive free radicals including nitrogen dioxide (•NO2), hydroxyl radical (•OH) and carbonate radical (•CO3). Each can readily induce LP within the phospholipid membranes of the mitochondrion leading to respiratory dysfunction, calcium buffering impairment, mitochondrial permeability transition and cell death. Validation of the role of LP in central nervous system secondary injury has been provided by the mitochondrial and neuroprotective effects of multiple antioxidant agents which are briefly reviewed.
Phenelzine (PZ) is a scavenger of the lipid peroxidation (LP)-derived reactive aldehyde 4-hydroxynonenal (4-HNE) due to its hydrazine functional group, which can covalently react with 4-HNE. In this study, we first examined the ability of PZ to prevent the respiratory depressant effects of 4-HNE on normal isolated brain cortical mitochondria. Second, in rats subjected to controlled cortical impact traumatic brain injury (CCI-TBI), we evaluated PZ (10 mg/kg subcutaneously at 15 minutes after CCI-TBI) to attenuate 3-hour post-TBI mitochondrial respiratory dysfunction, and in separate animals, to improve cortical tissue sparing at 14 days. While 4-HNE exposure inhibited mitochondrial complex I and II respiration in a concentration-dependent manner, pretreatment with equimolar concentrations of PZ antagonized these effects. Western blot analysis demonstrated a PZ decrease in 4-HNE in mitochondrial proteins. Mitochondria isolated from peri-contusional brain tissue of CCI-TBI rats treated with vehicle at 15 minutes after injury showed a 37% decrease in the respiratory control ratio (RCR) relative to noninjured mitochondria. In PZ-treated rats, RCR suppression was prevented (Po0.05 versus vehicle). In another cohort, PZ administration increased spared cortical tissue from 86% to 97% (Po0.03). These results suggest that PZ's neuroprotective effect is due to mitochondrial protection by scavenging of LP-derived 4-HNE.
The transcription factor NF-E2-related factor 2 (Nrf2) mediates transcription of antioxidant/cytoprotective genes by binding to the antioxidant response element (ARE) within DNA. Upregulation of these genes constitutes a pleiotropic cytoprotective-defense pathway which has been shown to produce neuroprotection in numerous models by decreasing lipid peroxidation (LP) as measured by the neurotoxic LP by-product 4-hyrdoxynonenal (4-HNE). As neuronal mitochondria have previously been shown to be susceptible to insult-induced LP-mediated oxidative damage, we sought to mechanistically investigate whether Nrf2-ARE activation in vivo could protect mitochondria from subsequent 4-HNE exposure ex vivo. Young adult male CF-1 mice were administered one of two known Nrf2-ARE activators as single I.P. doses – sulforaphane (SFP; 5.0 mg/kg) or carnosic acid (CA; 1.0mg/kg) – or their respective vehicles 48 hours prior to Ficoll isolation of rat cerebral cortical mitochondria. Purified mitochondria were then exposed ex vivo to 4-HNE for 15 minutes at 37°C which we showed to cause a concentration-related inhibition of mitochondrial respiration together with covalent binding of 4-HNE to mitochondrial proteins. We chose a 30 μM concentration of 4-HNE, which produced an approximate 50% inhibition of complex I or complex II-driven respiration, to assess whether prior in vivo the Nrf2-ARE activating compounds would increase the resistance of the isolated cortical mitochondria to 4-HNE's mito-toxic effects. Administration of either compound significantly increased (p< 0.05) expression of heme oxygenase-1 mRNA in cortical tissue 48 hours post-administration, verifying that both compounds were capable of inducing the Nrf2-ARE pathway. Moreover, the prior in vivo administration of sulforaphane (SFP) and carnosic acid (CA) significantly (p< 0.05) attenuated 4-HNE-induced inhibition of mitochondrial respiration for complex I while only carnosic acid acted to protect complex II. Furthermore, both CA and SFP significantly (p< 0.05) reduced the amount of 4-HNE bound to mitochondria proteins as determined by Western blot. These results demonstrate the capability of Nrf2-ARE induction in vivo to protect from 4-HNE toxicity to cortical mitochondria ex vivo. Ongoing studies will determine the therapeutic efficacy of Nrf2-ARE activators to attenuate traumatic brain injury induced pathophysiology.
J. Neurochem. (2011) 117, 579–588. Abstract Free radical‐induced lipid peroxidation (LP) is critical in the evolution of secondary injury following traumatic brain injury (TBI). Previous studies in our laboratory demonstrated that U‐83836E, a potent LP inhibitor, can reduce post‐TBI LP along with an improved maintenance of mouse cortical mitochondrial bioenergetics and calcium (Ca2+) buffering following severe (1.0 mm; 3.5 m/s) controlled cortical impact TBI (CCI‐TBI). Based upon this preservation of a major Ca2+ homeostatic mechanism, we have now performed dose‐response and therapeutic window analyses of the ability of U‐83836E to reduce post‐traumatic calpain‐mediated cytoskeletal (α‐spectrin) proteolysis in ipsilateral cortical homogenates at its 24 h post‐TBI peak. In the dose‐response analysis, mice were treated with a single i.v. dose of vehicle or U‐83836E (0.1, 0.3, 1.3, 3.0, 10.0 or 30.0 mg/kg) at 15 min after injury. U‐83836E produced a dose‐related attenuation of α‐spectrin degradation with the maximal decrease being achieved at 3.0 mg/kg. Next, the therapeutic window was tested by delaying the single 3 mg/kg i.v. dose from 15 min post‐injury out to 1, 3, 6 or 12 h. No reduction in α‐spectrin degradation was observed when the treatment delay was 1 h or longer. However, in a third experiment, we re‐examined the window with repeated U‐83836E dosing (3.0 mg/kg i.v. followed by 10 mg/kg i.p. maintenance doses at 1 and 3 h after the initial i.v. dose) which significantly reduced 24 h α‐α‐spectrin degradation even when treatment initiation was withheld until 12 h post‐TBI. These results demonstrate the relationship between post‐TBI LP, disruptions in neuronal Ca2+ homeostasis and calpain‐mediated cytoskeletal damage.
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